The aim of this proposal is to elucidate how different oncogenic pathways are wired to the cell cycle machinery in human mammary epithelial cells. The cell cycle machinery, in particular cyclins D1, D2 and D3 are the ultimate recipients of oncogenic signals. Emerging evidence indicates that the wiring of the oncogenic pathways to the cell cycle machinery is cell type specific. We recently demonstrated that mice lacking cyclin D1 are completely resistant to mammary carcinomas driven by the Ras and c-ErbB-2 oncogenes, while being fully susceptible to breast cancers driven by the c-Myc and Wnt-1 oncogenes. Our analyses revealed that in mammary epithelial cells (but not in several other cell types) the Ras and c-ErbB-2 are wired to the cell cycle machinery exclusively via cyclin D1, explaining the absolute dependency of these two oncogenes on D1. In contrast, the c-Myc and Wnt-1 can signal through other (as yet unidentified) targets. Two important issues remain unresolved: (1) Are the Ras and c-ErbB-2 also wired through cyclin D1 in human mammary epithelial cells? Resolution of this issue is very important, since our work in mice raises a possibility of novel, potentially highly specific therapies for human breast cancers, centered on cyclin D 1 inhibition. (2) How are other oncogenes, namely c-Myc and Wnt-1 wired to the cell cycle machinery in mammary epithelial cells? We will address these questions using a three-dimensional (3D) mammary epithelial culture system, developed by another member of the Program, Dr. Brugge. In this system, human non-transformed mammary epithelial cells can be genetically modified (by introducing activated oncogenes of viruses encoding small interfering RNA) and later grown in Matrigel, where the cells form differentiated acini. Dr. Brugge demonstrated that expression of several oncogenes in these cells leads to hyperproliferative changes in 3D cultures. We will use this system to identify cell cycle targets of the Ras, c-ErbB-2, Myc and Wnt-I in human mammary epithelial cells. We will then ask whether interfering with these targets using siRNA approach blocks the ability of the four oncogenes to drive hyperproliferation. Lastly, we will define molecular pathways leading from these oncogenes to the cell cycle machinery. The Specific Aims are to determine how the Ras and Neu oncogenes (Aim 1) as well as c-Myc and Wnt-1 oncogenes (Aim 2) are wired to the cell cycle machinery in human mammary epithelial cells.